MODEL FOR GONOCOCCAL INFECTION

Abstract: An animal having a disseminated infection with an organism requiring human factor C1q binding for pathogenicity. The animal, preferably a rat, is produced by administration of the organism coated with human factor C1q. The organism is, in particular, Neisseria gonorrhoeae. The rat infected with C1q-coated Neisseria gonorrhoeae provides a model for the disease state. This rat model allows the development and testing of possible prophylactic and therapeutic agents for the disease and is particularly useful as a model for the advanced stages of gonococcal infection in females. Prophylactic or therapeutic compositions including antibodies or Neisseria gonorrhoeae peptides with binding specificity for human factor C1q are described. A C1q synthetic analog having binding specificity for N. gonorrhoeae and without binding specificity for eukaryotic cells is described as being useful to treat gonococcal infection.

Description: MODELS FOR GONOCOCCAL INFECTION FIELD OF THE INVENTION The present invention is generally related to developing an animal model having an infection with an organism requiring human factor Clq binding for pathogenicity. More particularly, the present invention is related to providing an in vitro and in vivo rat model for testing prophylactic and therapeutic agents for gonorrhea and gonorrhea related disease states. BACKGROUND OF THE INVENTION Neisseria gonorrhoeae (GC) is an obligate human pathogen. Gonorrhoea is currently the most frequently reported bacterial infectious disease in the United States (about 2 million cases annually) (6) . Gonorrhoea is a sexually transmitted disease of the young population of reproductive age. Approximately 15% of patients with gonorrhoea develop severe complications and sequelae such as, pelvic inflammatory disease (PID) , infertility due to fallopian tube adhesion, ectopic pregnancy, and epididymitis. Frequent complications of PID are perihepatitis and peritonitis. Peritonitis may occur due to the menstrual blood refluxes through the fallopian tubes to the peritoneal cavity (7,8). Clinical observations strongly suggest that the peritoneal route is a dominating one in disseminated gonococcal infection (DGI) in women. Dissemination of N. gonorrhoeae from local peritonitis to the blood stream following pelvic inflammatory disease occurs in a significant portion of cases of gonorrhoea (8) . Association between pelvic pain and menstruation in PID may suggest that N. gonorrhoeae ascend at the time of the menses (containing Clq) and enters the peritoneal cavity. Approximately 75% of individuals who develop disseminated gonococcal infection are women. DGI is typically characterized by arthritis- dermatitis and/or endocarditis, yopericarditis, hepatitis, with functional abnormalities of the liver, and meningitis. Approximately 10% of pregnant women in many developing countries may be infected with gonorrhoea (9) . Gonorrhoea during pregnancy is a risk factor for premature delivery and may be transmitted to the neonate, thus causing ophthalmic neonatorum. Gonococcal ophthalmic neonatorum may be lead to blindness. There is an apparent lack of protective immunity following natural infection, even worse, a history of previous gonorrhoea is a risk factor for gonorrhoea. Therefore, development of a gonococcal vaccine is needed for the prevention of millions of cases in young people annually, including at least two million young Americans, and may result in the savings of millions of U.S. dollars. Years of previous studies suggest that it is almost impossible to develop successful gonococcal vaccine in the absence of an experimental animal model. Previous experiments suggest that a component of human blood glycoprotein Clq may be a host factor that increases attachment of GC to tissues and cells in vitro (10,11). Clq is a glycoprotein, structurally similar to collagen in terms of protein and carbohydrate moieties (12) . As a first component of complement, Clq present in human blood initiates activation of the classical pathway in the presence of antibody as well as in an antibody independent manner. Clq is synthesized by epithelial cells from representative portions of the entire human genitourinary tract. This correlates with the observation that urogenital epithelium from human and chimpanzees are the only species susceptible to gonococcal adherence in vitro. Development of an animal model of gonorrhoea has been hampered because only the human develops persistent colonization and infection. Since the gonococcal phenotype is influenced, not only by the organism itself, but also by the host, it seems to be important to preserve these interrelationships. This would require continued development and evaluation of models that mimic human gonococcal infection. Gonococcal urethritis has been produced experimentally in male chimpanzees (46) . Attempts to infect the genital tract of other animal species, including baboons, pig-tailed macaques, rhesus monkeys, squirrel monkeys, owl monkeys, capuchin monkeys, guinea pigs, and different strains of mice (47,48) have been unsuccessful. In addition, inoculation of gonococci into either the respiratory tract or mammary gland failed to produce infection (49). Kita et al . , (1981) reported gonococcal infection of the genital tract of mice of the ddY strain (112) . This strain from this single report is unavailable , no one has been able to use these animals for infection. Johnson et al . , (1989) reported that mice of the strains C3H, CBA, BALB/C, TO and ICR are resistant to gonococcal infection. Models on other animals, including implanted chambers were also proposed but were limited to the study of GC im unobiology (50,51,52,53,54,55,56). The experimental model for other related species N. meningitidis was developed on newborn rats. Meningococci survived only for six hours. Despite the short time of survival, this model allowed others to study the vaccine (57) . SUMMARY OF THE INVENTION The present invention involves provision of an animal having an infection with an organism requiring complement Clq binding for effective pathogenicity. The animal is produced by administration of the organism combined or coated with human factor Clq. The animal is normally resistant to the organism in question or experiences infection with only low frequency. The animal is, however, more susceptible to infection when the administered organism is combined or coated with human factor Clq. The animal may be a human or chimpanzee, for example, human male volunteers and chimpanzees normally experience a low rate and degree of infection. Preferably, however, the animal is non- primate, in particular, the most preferred animal is a rat. The organism may be a microbe such as bacteria or fungi or may be viral. In particular, the bacteria may be N. gonorrhoeae, Treponema pallidum or any gram negative bacteria that produce cardiolipin. The bacteria may be Chlamydia . A viral organism may be Human Immunodeficiency Virus having a protein which binds Clq. By "coated" we mean the organism is preincubated with Clq or, alternatively, Clq is administered first and the organism is administered in a later, separate, injection. This animal model is useful for the disease states caused by gonorrhea infection included pelvic inflammatory disease and disseminated gonococcal infection. The present invention also provides a prophylactic or therapeutic composition for intravaginal use which includes an antibody or N. gonorrhoeae peptides having binding specificity for human factor Clq. The composition is useful to prevent or treat gonococcal infection and may be in the form of a salve, douche, diaphragm jelly or an emollient. The N. gonorrhoeae peptides may be outer membrane peptides such as those having approximate molecular weights of 17kDa, 31kDa, 35kDa, and 48kDa. A further embodiment of the present invention is a method of developing a treatment regimen for gonococcal infection. This method includes infecting non-primate animals with N. gonorrhoeae coated with Clq and subjecting the animals to a test therapy while monitoring viable N. gonorrhoeae cells in the animal. The treatment regimen may involve the use of a therapeutic drug such as an antibiotic, for example. A preferred embodiment of the present invention is a method for formulating a vaccine for protection against gonococcal infection. The method includes the steps of i) administering gonococcal antigens to an animal, ii) infecting the animal with Clq coated N. gonorrhoeae , and iii) monitoring viable N. gonorrhoeae cells developing in said animal to determine an antigen resulting in inhibited developments of N. gonorrhoeae cells. The gonococcal antigens may be outer membrane peptides such as those having approximate molecular weights of 17kDa, 31kDa, 35kDa and 48kDa. The present invention provides for a prophylactic or therapeutic composition for intravaginal use including a Clq synthetic analog having binding specificity for N. gonorrhoeae and without binding specificity for eukaryotic cells. The composition is useful to prevent or treat gonococcal infection and may be in the form of a douche, diaphragm jelly or an emollient. Clq synthetic analogs may be analogs or fragments of Clq retaining binding specificity for N. gonorrhoeae but lacking the binding specificity for the eukaryotic host. One skilled in the art understands that a coating of Clq analog on N. gonorrhoeae would prevent native Clq from binding and would, thereby, decrease N. gonorrhoeae pathogenicity. Such an analog would be most effectively used as a topical agent similar to the administration of anti-Clq antibody. A further embodiment of the present invention is a method of treatment for gonococcal infection comprising the administration of a Clq synthetic analog having binding specificity for N. gonorrhoeae and without binding specificity for eukaryotic cells. Likewise any way of inhibiting the bridging function of Clq for tissue and GC is now taught to be therapeutic and/or prophylactic. One skilled in the art understands that the animal model of the present invention may provide a diagnostic method for using polymerase chain reaction (PCR) with appropriate primers to detect gonococcal cellular DNA in blood. Current methods exist for using PCR to detect GC in urine and vaginal fluid, however, methods do not exist for PCR detection of GC in blood, cerebrospinal fluid, joint fluid or amniotic fluid. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the dose dependent binding of Clq to N. gonorrhoeae JCl. Figure 2 shows that gonococcal attachment to leukocytes is enhanced by complement Clq. Figure 3 is a hypothetical model of attachment of W. gonorrhoeae to human tissue in the presence of Clq. Figures 4A and 4B show the attachment of N. gonorrhoeae JCl to ovarian tissue in the presence of Clq (A) and in the absence of Clq (B) . Notice in 4(B), lack of attachment. Figure 5 shows a Western blot of outer membrane protein of N. gonorrhoeae JCl probed with human Clq (line A) and with anti-cardiolipin IgG (line B) . Molecular mass standards in kilodaltons (on the left) . Solid circles show common bands of OMP reacted with complement- Clq as well as with anti-CL IgG. Figure 6 shows common and type specific structure of phospholipids tested for Clq deposition and anti-CL IgG binding; arrow indicates cleavage site for phospholipase C. Figure 7 shows the protective effect of anti-Clq IgG on the development of gonococcal bacteremia on newborn rats. Clq coated GC were used for i.p. inoculation. Figures 8A-8C relate to the effect of Clq on the attachment of Neisseria gonorrhoeae to the genital tissues. Clq-enhanced attachment of GC to rat and human tissues but not to that of rabbits and only a lesser extend in mice is shown by number of GC binding per standard unit area at 400 x magnification (8A) . The mean values and standard errors are shown for each group of data. Schematic diagrams (Figures 8B and 8C) represent proposed mechanisms. Figures 9A-9C relate to the Clq protection of GC from the bactericidal effect of newborn rat serum (in vitro) . Clq dose-dependent protection from the killing effect of newborn rat serum on GC was represented by the number of CFU per ml. Each bar represents the mean value of 3-5 independent experiments and standard error values for each group of data (Figure 9A) . Schematic diagrams representing proposed mechanisms are shown in Figures 9B and 9C. Figure 10 shows the inhibitory effect of anti-Clq- IgG on the development of gonococcal bacteremia. The dose-dependent protective effect of monospecific anti- Clq-IgG was represented by number of CFU per ml of tested blood. Protection observed at dilution 1:400 was statistically significant. (p< . 0.05). Figures 11A and 11B relate to the effect of Clq on the attachment of Neisseria gonorrhoeae to the human PMNs. Figure 12 shows the inhibitory effect of anti-Clq- IgG on the attachment of GC to PMNs. Open square D attachment of Clq treated GC to PMNs in presence of antibodies. Closed square ■ attachment of GC to Clq treated PMNs in presence of antibodies. The dose- dependent protective effect of monospecific anti-Clq-IgG was represented by number of GC per PMNs. Protection observed at dilution 1:500 was statistically significant (p<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides an animal model for testing vaccines and therapeutic agents against gonorrhea. The model, in a preferred embodiment, comprises live rats methodically infected with N. gonorrhoeae coated with human factor Clq. This rat model has less limitations than the human male volunteer model whose most severe limitation is the fact that the infection is localized to the male urethra. The rat model of the present invention allows the use of females to study the pathogenesis of DGI and PID, the most serious consequences of gonorrhea. The routes of entry and dissemination are equivalent to that in man. Clq coated N. gonorrhoeae is pathogenic for this animal model, the model is inexpensive and the illness is reproducible. Although N. gonorrhoeae strain JCl is primarily used in the provided examples, three additional Neisseria gonorrhoeae strains isolated from patients with gonococcal infection (from OB/GYN Clinic, UTMB, Galveston, TX) were used on our new experimental animal model. All of them developed gonococcal bacteremia similarly to strain JCl. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art. EXAMPLE I IDENTIFICATION OF Clq-BINDING MOLECULES IN OUTER MEMBRANES OF NEISSERIA GONORRHOEAE Gonococcal strains appear to deposit and activate complement, however, such strains survive in the human host. The exact molecules on which strains of N. gonorrhoeae from disseminated gonococcal infection deposit complement were heretofore unknown. Neisseria gonorrhoeae JCl was isolated from the blood of a female patient. Deposition of Clq on bacterial cells or purified outer membrane proteins of JCl was analyzed. Human Clq reacted with four polypeptides of approximate molecular weights 17kDa, 31kDa, 35kDa and 48kDa. Three bands (17kDa, 31kDa, 35kDa) which bind Clq reacted with anti-cardiolipin IgG. The reactivity of outer membrane proteins with Clq and anti-cardiolipin IgG was abolished after phospholipase C treatment. This example demonstrates that gonococcal outer membrane peptides serve as a binding target for human Clq. The antibody independent binding of Clq appears to take place on cardiolipin molecules associate with such peptides. Materials and Methods Patients, Bacteria and Growth Conditions N. gonorrhoeae JCl was isolated from the blood of a female patient with disseminated gonococcal infection. Strain JCl was resistant to killing by normal human serum in vivo and in vitro (101) . Strain JCl was grown on gonococcal base agar with Kellogg supplements GCK (Difco Laboratories, Detroit, Michigan) in 5% C0 2 at 37°C (102) . Human Serum Whole human blood was obtained by venipuncture from healthy donors who had no history of gonorrhoea. The blood was allowed to coagulate for one hour at room temperature, and then centrifuged for 5 minutes at 2,500 x g. The serum was then divided and stored in plastic tubes at -70°C. Serum Bactericidal Assays The serum bactericidal assay utilized was a modification of the procedure described previously (103,104). To determine the resistance of strain JCl to complement mediated killing by normal human serum, gonococci were grown for 18 - 20 hours on gonococcal based agar, then suspended in gonococcal base broth to an optical density at 595 nm of 0.2. A 1:1000 dilution was made, and the gonococcal dilution was incubated with decomplemented normal human serum (heated for 30 minutes to 56°C) at 37°C for 15 minutes. Then normal human serum was added to a final concentration of 25% and incubation was continued for 30 minutes at 37°C. From the assay mixture, 10-fold and 100-fold dilutions were made, and 50 μl was spread over the gonococcal base agar. The numbers of colony forming units (CFU) were determined after overnight incubation at 37°C in 5% C0 2 . Outer MftWh-fine Preparation (OMP) Cells of N. Gonorrhoeae JCl grown for 20 hours were removed from the plates, washed in 50 Mm Tris hydrochloride buffer (pH 8.0), and suspended in 20% sucrose in the same buffer. Lysozyme in a concentration of 1 mg per ml was added and the cells incubated for 30 minutes at 4°C. The cells were diluted 1:5 in 50 mM Tris hydrochloride buffer (pH 8.0), and were subjected to sonication in two bursts of 30 seconds each. Cellular debris was removed by centrifugation at 5,000 x g for 10 minutes, and gonococcal membranes were isolated by centrifugation at 100,000 x g for 1 hour. Outer membranes were isolated by differential solubilization in 1% sodium lauryl sarcosinate and centrifuged at 100,000 x g for 1 hour. The pellet contained the outer membrane proteins were washed with ethanol and suspended in sterile water (105) . Peptide patterns of purified OMP was clearly different from that of CMP. Protein I, II and III characteristic for OMP were present in the OMP preparation (not shown) . Polyacrylamide Gel Electrophoresis and Transfer of Fractionated Proteins to Nylon Membrane The outer membrane protein fraction of N. gonorrhoeae JCl was incubated at 100° in the presence of 1.5% sodium dodecyl sulfate (SDS) and 2.5% 2- mercaptoethanol for 2, 5, and 10 minutes, layered onto 5% polyacrylamide stacking gels, and fractionated in 12% polyacrylamide in the presence of 0.1% SDS (106). Two hundred ug of outer membrane proteins was added to each gel. Polyacrylamide gel electrophoresis was performed at room temperature using 30 mA per slab gel. Immunological characterization of the fractions was accomplished after transfer by electrophoresis at 150 mA for 1-1/2 hours at 22°C to a nylon membrane (107) . Reagents Human isolated Clq and polyclonal goat antisera to human Clq (Cytotech, San Diego, CA) were used. Horseradish peroxidase-conjugated rabbit anti-goat IgG, phospholipase C and phospholipids; phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and diphosphatidylglycerol (cardiolipin) , (Sigma Chemical, St. Louis, MO) were also utilized as reagents. Identification of complement (Clq) binding macromolecules in outer membrane proteins of N. gonorrhoeae was performed utilizing nylon membrane strips. They were incubated for 2 hours at 37° in 10% human serum albumin (HSA) diluted in veronal buffer saline (VBS) to block nonspecific protein binding sites. Subsequently, the nylon membrane strips were incubated with 70 ug/ml of isolated Clq diluted in VBS with 15 x 10 * 5 M Mg 2+ for 1 hour at 37°. The concentration of Clq in human serum is about 70 ug/ml. Localization of human Clq in specific zones in these strips was demonstrated with horseradish peroxidase-conjugated polyclonal goat antisera to human Clq. After three additional washes, localization of the peroxidase enzyme was visualized with 3.3'-diaminobenzidine in the presence of appropriate concentrations of hydrogen peroxide. The nylon membrane strips were washed three times for 15 minutes each between steps in 0.1% HSA in VBS in room temperature. To identify molecules that might cause nonspecific binding of these antibodies, control nylon membrane replicas were incubated with human Clq which had been heat inactivated at 56° for 30 minutes. Identification of anti-cardiolipin (anti-CL) IgG binding macromolecules in the outer membrane proteins of N. gonorrhoeae was performed using nylon membrane replicas of polyacrylamide gel (PAGE) . They were incubated with rabbit anti-cardiolipin IgG (1:300) diluted in 0.1% HSA in VBS with 0.05% Tween 20 in room temperature. Before exposure to these antisera, the nylon membrane strips were incubated 2 hours in 37°C in 10% HSA diluted in VBS to block nonspecific binding. The localization of antibodies to specific components in the transblots were identified using horseradish peroxidase- conjugated goat anti-rabbit IgG in a dilution of 1:300. Figure 5 shows a Western blot of outer membrane protein of N. gonorrhoeae JCl probed with human Clq (line A) and with anti-cardiolipin IgG (line B) . Molecular mass standards in kilodaltons (on the left) . Solid circles show common bands of OMP reacted with complement-Clq as well as with anti-CL IgG. Phospholipase C Treatment Nylon membrane replicas of PAGE gels or nylon membrane dots were incubated for 2 hours with phospholipase C (0.5 units/ml) diluted in 1% BSA Tris saline pH 7.3 activated with 0.001 M CaCl 2 . Before exposure to human Clq, the nylon membrane strips were washed three times for 15 minutes each in 0.2% sodium dodecyl sulfate (to stop reaction) , and three times in 0.1% HSA in VBS, and incubated 2 hours in 37°C with 10% HSA in VBS to block nonspecific binding. Control nylon membrane replicas or dot blots were incubated without phospholipase C. staininσ of GC Gels; Binding of Clq to GC cells was done by indirect fluorescence using as first antibody goat anti-Clq-IgG and second fluorochrome conjugated rabbit anti-goat IgG. GC cells were also immobilized on nylon membranes and stained according to the procedure used for OMP staining as described above. Results & Discussion In this Example, the deposition of Clq on intact bacterial cells was tested by two alternative approaches. First GC cells were incubated with Clq and immunostained with anti-Clq rabbit IgG and a second antibody to rabbit IgG conjugated with fluorochrome. Staining of cells incubated with Clq was found in the fluorescence microscope. In the second approach, dot blot analysis of whole cells immobilized on nitrocellulose membrane was performed. Strong staining was also observed by this method indicating that molecules present on the bacterial surface binds Clq without an antibody intermediary (Table D- Table 1. Reactivity of GC cells and purified OMP** with Clq and anti-CL IgG Reactivity Before PLC* Treatment After PLC Treatment with Clq Anti-CL Clq Anti-CL IgG IgG GC Cells + + NT NT OMP + + Note: (+) indicates positive reaction (-) indicates no reaction * PLC = phospholipase C NT = not tested ** OMP = GC outer membrane preparation Deposition of complement Clq on purified outer membrane proteins of N. gonorrhoeae JCl immobilized on nylon membrane was tested by dot blot analysis. Antibody against Clq reacted with immobilized proteins that were exposed to Clq (Table 1) . Specific molecules which exhibited this function were identified in SDS-PAGE fractionated outer membrane proteins of JCl strain, which were transferred to a nylon membrane incubated with human Clq. Antibody against human Clq reacted with four polypeptides including 17kDa, 31kDa, 35kDa, and 48kDa (Fig. IA) . Our studies on human mitochondria proteins showed that a number of proteins are associated with cardiolipin. The peptide cardiolipin complexes of mitochondria origin were found to bind Clq (108) . Therefore, antibody against cardiolipin was utilized to determine if the outer membrane-preparation of N. gonorrhoeae JCl contained diphosphatidylglycerol (cardiolipin) . Table 1 shows the results of a dot blot of outer membrane protein of N. gonorrhoeae JCl probed with anti-cardiolipin IgG. Anti-cardiolipin IgG reacted with outer membrane proteins of JCl. Further analysis of cardiolipin-containing molecules in the outer membrane proteins of N. gonorrhoeae , were performed on resolved proteins transferred to nylon membranes and probed with rabbit anti-cardiolipin IgG. Six molecules having estimated molecular weights of 17kDa, 28kDa, 31kDa, 35kDa, 50kDa and 55kDa were detected by anti-cardiolipin IgG (Fig. IB) . Three bands (17kDa, 31kDa, 35kDa) which bind Clq reacted with anti-cardiolipin IgG. Anti- cardiolipin IgG recognized three additional molecules (28kDa, 50kDa, 55kDa) which did not bind Clq. In order to test if the anti-cardiolipin IgG utilized in these experiments, bind specifically to cardiolipin, the nylon membrane dot blots of purified selected phospholipids were incubated with anti-cardiolipin IgG (Table 2) . Five different concentrations were used for all phospholipids (5 mg, 10 mg, 20 mg, 40 mg, 80 mg per 1 ml) and 5 different dilutions of anti-cardiolipin IgG (1:50; 1:100; 1:200; 1:300; 1:500). Anti-cardiolipin IgG reacted with cardiolipin, but did not react with phosphatidylethanolamine, phosphatidylinositol, or phosphatidylserine (Table 2 and see structures of phospholipids in Fig. 6) . Table 2. Reactivity of selected phospholipids with isolated human Clq and with anti-cardiolipin IgG (anti-CL IgG) Phospholipid Before PLC* After PLC Treatment Treatment Clq Anti-Cl Clq Anti-CL IgG IgG Phosphatidylethanol¬ amine Phosphatidylinositol Phosphatidylserine Diphosphatidylglycerol Note: (+) indicates positive reaction (-) indicates no reaction * PLC = phospholipase C Purified cardiolipin and dot blots of outer membrane proteins of N. gonorrhoeae JCl that were treated with phospholipase C lost reactivity with Clq and anti- cardiolipin IgG (Tables 1 and 2) . Further investigation of molecules affected by phospholipase C treatment was performed on nylon membrane replicas of separated outer membrane proteins incubated with the enzyme for 3 hours at 37°C. In control experiments phospholipase C was replaced with phosphate buffered saline. The reactivity of all molecules of outer membrane proteins with Clq and anti-cardiolipin IgG was abolished. Although some bacterial species have been suggested to bind Clq without an antibody intermediary, there has been no such phenomenon described for N. gonorrhoeae . Moreover, gonococcal macromolecules involved in Clq binding have not been identified yet. Such molecules, if identified, might be of potential importance to understand and prevent disorders associated with gonococcal infections. This example demonstrates that gonococcal cells and isolated outer membrane peptides may serve as a binding target for human Clq. Attachment of Clq to at least four peptides occurred without antibody intermediary. The antibody independent binding of Clq appears to take place on cardiolipin or cardiolipin-like molecules associated with some OM peptides. The few outer membrane- cardiolipin-like molecules on N. gonorrhoeae, which reacted with anti-cardiolipin IgG, did not bind Clq. This may be explained by Clq activation tests with vesicles containing increasing amounts of cardiolipin indicated that over 30 mol % of cardiolipin is required to activate Clq (109) . The majority of previous studies on complement binding and activation on bacterial surface used an approach that included incubation of intact cells with complement, solubilization of cells and separation by SDS-PAGE electrophoresis. With this approach however, solubilization and SDS treatment may have resulted in dissociation of bound molecules, so that obtained results may be difficult for interpretation. In our study we have first analyzed Clq binding to the surface of intact cells. Once binding was confirmed, intact complement components were used for identification of potential binding sites on bacterial outer membrane peptides separated on SDS-PAGE and immobilized on nitrocellulose or nylon membrane. Although the solubilization process of OMP in SDS was performed for 2, 5 and 10 minutes, Clq binding to several OMP was not abolished. Recent findings demonstrate that peptide-cardiolipin molecules are present in mitochondria and are resistant to SDS page treatment (108) . Since cardiolipin is known to be present in gonococcal cells, consequently we considered the possibility that phospholipid like molecules, such as cardiolipin, may be involved in Clq deposition. Such molecules are found to exist in association with some OM peptides. Anti-cardiolipin antibody used in the present study shows high specificity only for diphosphatidylglycerol (cardiolipin) . Diphosphatidylglycerol, one of the cell membrane phospholipids unique in its structure and function, possess two negatively charged phosphate group. In contrast, the other phospholipids carry only one negatively charged phosphate groups. Results demonstrated that among tested phospholipids only cardiolipin may deposit Clq. This finding is in agreement with other investigations which demonstrated that Clq binding on cardiolipin may initiate deposition and activation of classical pathway (108,109). Taken together, applicants suggest that the nondenatured cardiolipin-peptides molecules on intact gonococcal cells may be able to bind Clq. In the light of the above presented observations we propose that also bacterial cells of N. gonorrhoeae may deposit and activate the complement cascade without a requirement for specific antibody. Gonococcal septicemia is associated with multiplication of bacterial cells in the bloodstream resulting in dissemination into several anatomical sites including joints, genitourinary tract and other tissues. Consequently applicants believe that circulating and tissue-associated bacterial cells may continuously activate the complement cascade without much harm to the pathogen. In turn, activated complement or individual C components may display their biological activities including increased permeability an infiltration by the cellular immune system. Activated humoral and cellular systems may lead to pathogenic sequelae such as arthritis, dermatitis or perhaps pelvic inflammatory disease, all devastating syndromes associated with gonococcal infections. EXAMPLE II GONOCOCCAL ATTACHMENT TO HUMAN LEUKOCYTES IS ENHANCED BY COMPLEMENT Clq Phagocytosis and intracellular survival of Neisseria gonorrhoeae in leukocytes appear to be an important feature of gonococcal strains associated with disseminated infections. Attachment of N. gonorrhoeae to human cells, including leukocytes, is recognized as an important first step necessary prior to invasion or phagocytosis. Example I describes gonococcal outer membrane receptors that bind purified Clq without opsonizing antibody. Clq receptors are known to be present on mammalian cells including leukocytes. Clq molecules mediate the enhancement of phagocytic cell functions. Strain of N. gonorrhoeae JCl from disseminated gonococcal infection (DGI) is resistant to killing by human defense mechanism. This strain was cultivated on GC medium and used for experiments (optical density of 0.2 at 600 nm) . Human PMNs were freshly isolated. Microscope slides of PMNs were prepared and stored at -70°C until used. Purified Clq (70 mg/ml) was preincubated either with bacteria or leukocytes. Washed bacteria and leukocytes on slides were then used for experiments. Binding of gonococci to leukocytes was estimated in phase-contrast microscope. Attachment of gonococcal cells to leukocytes pretreated with Clq was observed (20-30 bacterial cells per leukocyte) (Fig. 2) . In the control fewer attached gonococcal cells (0-4 per leukocyte) were observed (Fig. 2) . Similarly gonococcal cells preincubated with Clq displayed increased binding to leukocytes (10-20 gonococcal cells per leukocytes) . The binding of gonococcal cells to its receptors on human leukocytes was inhibited by monospecific antibody against Clq (Fig. 2) . Clq, therefore, is a host factor that increases attachment of N. gonorrhoeae JCl to human leukocytes (Fig. 3) . Therefore, increased binding via Clq should be considered as a virulence factor of N. gonorrhoeae in DGI. In preliminary studies on animal tissues, the present inventors have tested attachment of N. gonorrhoeae JCl to various genital tissues from different animal species in the presence and absence of Clq and observed Clq-dependent binding of GC to the tissues from rats (Table 3) . GC did not attach very well to the various animal tissues from other tested species (Table 3). Table 3: Attachment of N. gonorrhoeae to different tissues using Clq as a potential adhesion. -(0); ±(1-4); +(5-10), ++(10-20); and +++(30-40) gonococcal cells per unit area. *Rabbit tissues kindly provided by V. Schnell, M.D., Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, Texas. EXAMPLE III GONOCOCCAL ATTACHMENT TO HUMAN OVARIAN TISSUE Attachment of N. gonorrhoeae to human cells is a necessary first step prior to colonization or invasion. The majority of gonococcal attachment studies have been performed on cells of human fallopian tubes. Very little is known about the attachment of gonococcal organisms to ovary cells, although ovarian tissue is often involved in the infectious process, resulting in, e.g., pelvic inflammatory disease (PID) and infertility. Outer membrane proteins and fimbriae of N. gonorrhoeae are known to play an important role in attachment to selected human tissues or cells, as observed by in vitro experiments (83,84). However, many tissues or cell types are not easily recognized by gonococcal cells. Therefore, there may be alternative attachment mechanisms (10) . The inventors'' experiments indicate that Clq serves as a bridging molecule between gonococcal cells and leukocytes. Clq, a heat-labile protein present in human blood and ovarian follicular fluid, is the first component of complement, initiating activation of the classical pathway. Clq is a glycoprotein that is structurally similar to collagen in terms of its protein and carbohydrate moieties. Clq is involved in different types of immunoregulatory functions (85) . The molecular mechanism associated with the bridging function of Clq may include the following phenomenon. These possible mechanisms are presented to more fully elucidate an understanding of the present invention's importance but should not be viewed to limit the appended claims. Clq appears to recognize receptors on gonococcal (GC) cells; the present inventors have found outer membrane peptides of GC that interact with Clq without opsonizing antibody (40) . Conversely, Clq appears to recognize receptors on leukocytes and, therefore, bridge the bacteria and the host cell (10) . Receptors for Clq are distributed in different tissues as well, therefore, Clq-promoted binding occurs also in other sites exposed to gonococcal infection. Cryostat sections of snap frozen human ovarian tissue were prepared and stored at -70°C until used. A suspension of strain of N. gonorrhoeae JCl isolated from female blood was prepared (optical density of 0.2 at 600 nm) . Cryostat sections were incubated with 40 μl of GC suspension in the presence or absence of Clq (20 μg/ml) for 30 min in a moist chamber. Samples were washed five times with PBS pH 7.2 and bacterial binding visualized in phase-contrast or fluorescence microscope. Figures 4A and 4B show attachment of N. gonorrhoeae JCl to ovarian tissue in the presence and absence of Clq. Attachment to the ovarian tissue is dramatically increased if either tissue or bacteria are preincubated with Clq. In the absence of Clq, only few bacterial cells were found to be attached to the ovary. The capacity of Clq binding to gonococcal cells immobilized on microtiter plates was investigated. Different concentrations of purified Clq and monospecific antibody (IgG) to Clq were used in an ELISA assay. Figure 1 shows a dose-dependent binding of Clq to N. gonorrhoeae JCl. Clq binding was saturated at about 8 μg/ml; concentration of Clq in human blood is about 70 μg/ml. In summary, these experiments showed that Clq is a host factor that increases attachment of N. gonorrhoeae to human ovarian tissue. This attachment may be explained by a bridging function of Clq that recognize receptors in both the bacteria and the tissue. Clq dependent attachment should result in an increased colonization of the human genital tissue such as ovary, especially when ovarian follicular fluid contains complement at concentration similar to the blood. Therefore, increased binding, via complement, should be considered as a possible virulence factor of N. gonorrhoeae in pelvic inflammatory disease or infertility. EXAMPLE IV GONOCOCCAL INFECTION IN NEWBORN RATS WITH Clq COATED GONOCOCCAL CELLS The present example describes a rat model of gonococcal infection. Bacteria and Growth Conditions N. gonorrhoeae JCl was isolated from the blood of a female patient with disseminated gonococcal infection. Strain JCl is resistant to killing by normal human serum in vivo and in vitro (36) . Strain JCl was grown on gonococcal base agar with Kellogg supplements GCK (Difco Laboratories, Detroit, Michigan) in 5% C0 2 at 37°C (36,45). A suspension of N. gonorrhoeae in veronal buffer saline (VBS) was prepared with an optical density of 0.8 at 595 nm, divided into two tubes and incubated for 30 min. in 5% C0 2 at 37°C; one tube in the presence and the other in absence of Clq (40 μg/ml) (Cytotech, San Diego, CA) . Gonococcal colonies were visualized with a dissecting microscope and were phenotyped by the method of Swanson, et al . , (61,62). The difference in size and color/opacity colony types (TI, T2-piliated and T3, T4 non-piliated in combination with 0p+ and Op-) was used separately for all experiments and the outcoming types quantitated. Animals and Experimental Infection Pregnant female Sprague-Dawley rats were housed under standard conditions (25°C; relative humidity 40%) on a 12:12 hours light to dark cycle and given food and water libitum. The rat pups remained with their mothers after parturition and were used at the age of three days (± 1 day) , when they weighed 10 to 15 g. For each experiment, about 5 pups from the same litter were used. The suspension of N. gonorrhoeae was intraperitoneally injected in 0.1 ml volume. After the injection, the pups were returned to their mothers. 10 x 10 8 , 5 x 10 8 , 1 x 10 8 , 5 x 10 7 , 1 x 10 7 and 5 x 10 6 bacterial cells per ml were used to select the lowest concentration that may develop infection and the concentration that may cause death of infected animals. The kinetics of bacteremia was followed by serial blood cultures. Recovery and Identification of Gonococci from Blood and Other Tissue of Infected Pups. Groups of pups were sacrificed after 1, 2, 5 and 6 days. Blood cultures were collected before sacrifice. Aliquots (0.05 ml) of blood were cultured on GC agar and in 1 ml of Columbia broth containing sucrose, sodium polyethane sulfonate, "increased cysteine," and C0 2 . Gonococci were identified by Gram stain, fluorescent antibody stain (no. 2361-56; FA-W. gonorrhoeae ; Difco, Detroit, Mich.), colony morphology, oxidase positivity, and sugar fermentation pattern. Characterization of colony types of N. gonorrhoeae isolated from different tissues were analyzed by the method of Swanson (61, 62) . Gram-methyl green-pyronin-light green stain, which was developed specifically for the purpose of detecting bacteria in tissue sections, was used for detection of GC cells as well (63) . Results Table 4 shows the susceptibility of newborn rats to gonococcal infection in the presence and absence of human complement Clq. N. gonorrhoeae was isolated on chocolate agar plates from the blood of newborn rats 48 hours after the injection of gonococcal cells coated with Clq. N. gonorrhoeae was not isolatable from blood of newborn rats injected with N. gonorrhoeae , uncoated with Clq. Table 4. Post-Inoculation recovery of N. gonorrhoeae from newborn rat blood. * Time N. gonorrhoeae N . gonorrhoeae coated with Clq Hours After Number Number of Number Number of Inoculations of Animals of Animals of GC Animals With Animals With Used Positive Used Positive Blood Blood Culture Culture 3 5 0 7 7 6 5 0 7 7 9 5 0 7 7 12 5 0 7 7 24 4 0 4 4 48 2 0 2 2 Total 26 0(0%) 34 34(100%) * Summary of three different experiments except for the forty-eight hour experiment which was performed once. The kinetics and localization of GC in other tissues during gonococcal infection in this rat model were tested in the next series of experiments. The results are shown in Table 5. Table 5. Post-inoculation recovery of Gonococcal Cells (GC) from newborn rat tissues. GC GC Treated With Clq C o l ony Time after Tissues Number of Animals CFU Number of Animals CFU/ml Type % Tl Inoculation Used Infected Used Infected Blood 2 0 0 2 2 10 4 100% ONE DAY CSF 2 0 0 2 2 10 3 100% Liver 2 0 0 2 2 2xl0 4 100% Heart 2 0 0 2 2 2xl0 3 100% Urine N.D. N.D. N.D 1 0 0 0 • Blood 2 0 0 2 2 2xl0 4 99% CSF 2 0 0 2 2 8xl0 2 100% TWO DAYS Liver 2 0 0 2 2 3xl0 4 100% Heart 1 0 0 2 2 2xl0 3 99% Blood 2 0 0 2 0 0 0 FIVE DAYS Liver 2 0 0 2 2 4xl0 4 100% Uterus 1 0 0 1 1 0.2x10 98% GC GC Treated With Clq C o lony Time after Tissues Number of Animals CFU Number of Animals CFU/ml Type % Tl Inoculation Blood 2 0 0 3 1 0.5x10 99% SIX DAYS CSF 2 0 0 3 0 0 100% Liver 2 0 0 3 2 4X10 3 100% 2X10 4 Heart 2 0 0 3 2 0.3x10 100% 2 ,o Uterus 1 0 0 2 1 0.4x10 99% 2 ,ND N. gonorrhoeae JCl (0.1 ml) in concentration of 5xl0 6 /ml was inoculated. Colony types Tl-85% and 0p(+) 80% were used for inoculation. GC isolated from all infected tissues showed in all animals one colony form (Tl) : small, domed, highlighted colonies which are known to produce pili (Swanson Tl) 98-100% of all colonies recover from newborn rat tissues were opaque (Op) . These results indicate that GC coated with human Clq may develop DGI in rat pups. Data were summarized from experiments on 17 pups from two litters. Tissues of 8 pups from one litter were analyzed on day one and day five after GC inoculation. Tissues of nine pups from another litter were analyzed on day one, two and day six after GC inoculation. Overall, GC coated with Clq injected i.p. were found to reach the blood stream and survive in pups blood up to 5-6 days (Table 5) . At day 6, GC could be cultured from some organs, e .g. , liver. Microscopic estimation of tissues changes indicated that the liver was enlarged, presumably from inflammation by gonococcal infection. Rat pups inoculated with Clq coated GC were less active, their skin showed increased redness and overall they looked ill as compared to the control pups. GC coated with Clq, circulating with blood may penetrate to the cerebrospinal fluid (CSF) , and reach different tissues. Clq may be a bridging molecule (in vivo) between GC cells and infected tissue. It is known that Clq binds to human tissues via Clq receptors (13,14,15,17,59) . In the control group, GC not coated with Clq did not develop any symptoms of gonococcal infection in the pups from the same litter. GC were not isolated from any pups, organs at any time. Several phenomena may have happened: 1) GC, not coated may not have reached the blood stream, or were killed immediately by the pups' serum and/or leukocytes, 2) GC, when not coated, may not attach to various tissues in vivo and 3) immune response in pups is efficient to clear infection with GC not coated with Clq. Results from Table 4 and Table 5 indicate that 100% of newborn rats were infected with N. gonorrhoeae pretreated with Clq. In control experiments, none of the newborn rats were infected with N. gonorrhoeae when Clq was not present. These results suggest that human serum glycoprotein Clq plays an important role in gonococcal infection and is necessary in the experimental gonococcal infection model on newborn rats. Isolation of GC cells from blood suggests that N. gonorrhoeae in the presence of human complement Clq becomes invasive in vivo and penetrates from the peritoneal cavity to the blood system of newborn rats. Clq coated GC circulated in blood stream may disseminate and reach other tissues and colonize them. The present inventors then investigated if anti-Clq IgG would prevent animals from infection. Dilutions of anti-Clq IgG (1:50, 1:100, 1:500) were given i.p. 1.5 hours before inoculation of Clq coated or non-coated GC cells. These experiments showed dose-dependent protection of newborn rats from GC-Clq promoted infection (Fig. 7). UTILITY OF THE RAT MODEL The availability of a system for infection of rats with N. gonorrhoeae can now serve as an experimental tool for the study of gonococcal infection and can be employed for numerous purposes, including those listed below. 1. The development of vaccines and other immunization procedures designed to prevent infection with N. gonorrhoeae and/or to prevent subsequent development of gonorrhea in infected humans (e.g., procedures designed to maximize neutralizing antibody production) . 2. Development and testing of prophylactic and therapeutic drugs and procedures including passive immunization with antibodies to prevent and combat gonorrhea in humans. 3. Testing various isolates of N. gonorrhoeae in order to determine whether there are significant differences in the ability of different strains of N. gonorrhoeae to cause disease. 4. To determine whether there are synergistic effects in gonorrhea infection caused by use of certain drugs or other substances. It is noted that, given the model system of the present invention, all of the above-mentioned utilities can be routinely accomplished without specific elaboration herein of the procedures involved, it being pointed out that such procedures are quite standard and well known to those of ordinary skill in the art to which this invention pertains. Thus, a method for testing anti-gonorrhea therapeutic agent, comprises (a) administering a therapeutic agent to be tested, to live N. gonorrhoeae infected rats; and (b) determining the effect of said agent on the progression disseminated of gonorrhea infection in said rats, an arresting of gonorrhea infection progression in said rats being indicative of the efficacy of said agent. Similarly, a method for testing anti-gonorrhea vaccine comprises (a) administering an anti-gonorrhea vaccine to be tested, to a group of live uninfected rats; and (b) then infecting the rats of step (a) with Clq coated N. gonorrhoeae while concomitantly infecting a control group of unvaccinated rats with the same batch of Clq coated N. gonorrhoeae , a failure of gonorrhea infection to develop in the rats of group (a) and the progression of gonorrhea infection in control group of rats being indicative of the efficacy of the anti- gonorrhea vaccine. EXAMPLE V Use of Anti-Clq Antibody or Clq Receptor Fragments Polyclonal antibodies against human Clq were prepared. Three rabbits were immunized subcutaneously with purified Clq protein (lOOμg) prepared in incomplete Freund's adjuvant (50μl) . The secondary immunization was given one week later, and the animals were bled a week later to test immune response. Subsequent boosts have been given once every week for four weeks (total 6 weeks) . Prior to the first injection, blood has been collected (2 mL) from each animal to obtain control serum levels of antibody to the injected preparation. Commercially available monospecific polyclonal goat antisera to human Clq was also obtained from Cytotech, San Diego, CA. Monoclonal anti-Clq antibodies that prevent the N. gonorrhoeae-tissue bridging function may also be used. Anti-human complement Clq monoclonal antibodies are available from the American Type Culture Collection, Rockville, Maryland, as ATCC HB 8327 and HB 8328. These hybridomas are defined in U.S. Patent 4,595,654, which is incorporated by reference herein. Experiments have now shown that Clq is a human factor that not only increased the virulence of gonococcal cells (GC) in vitro by mediating GC attachment (Figure 8) , but also by increasing GC resistance to bacteriolytic effect of serum components (Figure 9) . These effects allowed survival and dissemination of GC in experimental animals. Detection of Clq on GC attached to human tissues taken from patients with gonococcal infection indicates involvement of Clq in pathogenesis of human gonococcal infection (Nowicki, et al . 1993). Attachment of GC to host tissues is a first and necessary step to establish gonococcal infection. Inhibition of GC attachment (the first step in pathogenesis) represents an important strategy in new prophylactic approaches. Anti-Clq antibodies reduced the virulence function of Clq-coated GC in vitro and in vivo. Figure 10 shows that anti-Clq antibodies protect animals from gonococcal infection by Clq-coated GC in a dose-dependent fashion, demonstrating a causal effect of Clq function. Anti-human Clq antibodies used in dilutions 1:800, 1:400, 1:200, 1:100, and 1:50 reduced the number of GC recovered from the blood to 10 3 , 10 2 , 10, and 0 CFU/ml respectively (Figure 10) . Blood from control animals without antibody treatment showed 10 5 CFU/ml (Figure 10) . Anti-Clq inhibits or even abolishes attachment of GC to human polymorphonuclear leukocytes (PMN) (Figure 11) and other tissues including endometrium and ovaries (not shown) . Figure 11A shows Clq-enhanced attachment of GC to PMNs is shown by the number of GC binding per PMNs. A) Binding of preincubated with Clq N. gonorrhoeae to human PMNs. B) Binding of N. gonorrhoeae to PMNs preincubated before with human Clq. C) Binding of preincubated with Clq N. gonorrhoeae to PMNs also preincubated with Clq. D) Binding of N. gonorrhoeae to PMNs in the absence of Clq. Values were determined in triplicate and error bars represent the standard deviation from the means. The binding of N. gonorrhoeae in presence of Clq was 10 to 30 fold higher than Clq- independent values. Schematic diagrams represent proposed mechanisms (Figure 11B) . Anti-Clq-IgG protects animals from experimental gonococcal infection. Anti-Clq-IgG abolishes attachment of GC to host tissues. Anti-Clq-IgG antibodies are useful in protection against gonococcal infection. Anti- Clq-IgG antibodies may be used in vaginal cream before intercourse (especially in groups with high risk for gonococcal infection) . GC attachment to host tissues that express Clq receptors is supported by Clq on the surface on GC. Therefore, to block attachment of GC to host tissues, synthetic analogues of Clq and/or Clq receptors expressed in host tissues and/or GC may be used. Figure 12 shows the inhibitory effect of anti-Clq- IgG on the attachment of GC to PMNs. Open squares D show attachment of Clq treated GC to PMNs in presence of antibodies. Closed squares ■ show attachment of GC to Clq treated PMNs in presence of antibodies. The dose- dependent protective effect of monospecific anti-Clq-IgG was represented by number of GC per PMN. Protection observed at dilution 1:500 was statistically significant (p<0.05) . 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Claims: CLAIMS 1. A rat having a disseminated gonococcal infection. 2. A rat having a disseminated gonococcal infection produced by administration of gonococci coated with human factor Clq. 3. A non-primate animal having a disseminated gonococcal infection induced by administration of factor Clq prior to or concomitant with Neisseria gonococci . 4. A rat having a disseminated gonococcal infection induced by administration of strain JCl N. gonorrhoeae pretreated with human factor Clq. 5. A rat model for pelvic inflammatory disease, said animal produced by the administration of N. gonorrhoeae coated with human factor Clq. 6. A prophylactic or therapeutic composition for intravaginal use comprising an antibody having binding specificity for human factor Clq, said composition being useful to prevent or treat gonococcal infection. 7. A prophylactic or therapeutic composition for intravaginal use comprising peptides derived from N. gonorrhoeae or Clq receptor protein and having binding specificity for human factor Clq, said composition being useful to prevent or treat gonococcal infection. 8. The composition of claim 7 wherein the peptides are outer membrane N. gonorrhoeae peptides having approximate molecular weights of 17kDa, 31kDa, 35kDa, or 48kDa. 9. The composition of claim 6 or 7 defined further as being in the form of a douche or salve. 10. The composition of claim 6 or 7 defined further as being in the form of a diaphragm jelly. 11. The composition of claim 6 or 7 defined further as being in the form of an emollient. 12. A method of developing a treatment regimen for gonococcal infection comprising infecting rats or mice with N. gonorrhoeae coated with Clq and subjecting said infected rats or mice to a test therapy while monitoring viable N. gonorrhoeae cells. 13. The method of claim 12 wherein the treatment regimen involves use of a therapeutic drug. 14. The method of claim 12 wherein the treatment regimen involves use of an antibiotic. 15. A method for formulating a vaccine for protection against gonococcal infection, the method comprising: administering gonococcal antigens to an animal; infecting said animal with Clq-coated N. gonorrhoeae; and monitoring viable N. gonorrhoeae cells developing in said animal to determine an antigen resulting in inhibited development of N. gonorrhoeae cells. 16. The method of claim 15 wherein the gonococcal antigens are outer membrane peptides having approximate molecular weights of 17kDa, 31kDa, 35kDa, or 48kDa. 17. A therapeutic composition for intravaginal use comprising a Clq analog or fragment having binding specificity for N. gonorrhoeae and being without binding specificity for eukaryotic cells, said composition being useful to treat gonococcal infection. 18. A Clq receptor protein or protein fragment having affinity for human Clq and inhibiting interaction of N. gonorrhoeae bearing Clq and tissue. 19. The method of claim 12 or 15 where the gonococcal infection is a disseminated infection or pelvic inflammatory disease.

Inventors: ANDERSON GARLAND D, MARTENS MARK G, NOWICKI BOGDAN, NOWICKI STELLA, ANDERSON, GARLAND, D., MARTENS, MARK, G., NOWICKI, BOGDAN, NOWICKI, STELLA
Applicants: ANDERSON GARLAND D, MARTENS MARK G, NOWICKI BOGDAN, NOWICKI STELLA, UNIV TEXAS, ANDERSON, GARLAND, D., BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM, MARTENS, MARK, G., NOWICKI, BOGDAN, NOWICKI, STELLA
Application year: 1994
Main classification: C12N15
IPC codes: A01K67, A61K38, A61K39, C07K14, C07K16